PINAR GOC RASGELE
1*, MERAL KEKECOGLU
2,
FULYA DILEK GOKALP MURANLI
31 Duzce University, Beekeeping Research Development and Application Center, 81620 Duzce, Turkey 2 Duzce University, Faculty of Science and Letters, Department of Biology, 81620 Duzce Turkey
3 Trakya University, Faculty of Science, Department of Biology, 2203, Edirne/Turkey
* Corresponding author`s e-mail: pinargocrasgele@gmail.com Keywords: Cobalt chloride, copper chloride, heavy metal, mice, micronucleus assay.
Abstract: The aim of our research was to investigate the genotoxic effects of cobalt chloride and copper chloride in mouse bone marrow cells using the micronucleus (MN) assay. The three different concentrations of cobalt chloride (11.2, 22.5 and 45 mg kg-1) and copper chloride (1.17, 2.35 and 4.70 mg kg-1) were injected intraperitoneally to mice for 24 and 48 hours. It was observed that both of these heavy metals induced a signifi cant increase in frequency of micronucleated polychromatic erythrocytes (MNPCE) at different concentrations in mice for 24 and 48 hours when compared with the control. Furthermore, the signifi cant reduction for the polychromatic erythrocyte/normochromatic erythrocyte (PCE/NCE) ratio which is indicative of bone marrow cytotoxicity was observed in bone marrow cells which were treated with copper chloride at all concentrations for 24 and 48 hours. No reduction of the PCE/NCE ratio was observed both 24 and 48 hours after all the doses of cobalt chloride tested as compared to the negative control. These results lead us to the conclusion that copper chloride may have genotoxic and cytotoxic properties due to induction in the frequency of MN and a reduction in PCE/NCE ratio in bone marrow cells of mice, whereas cobalt chloride induced only genotoxic effect in mice bone marrow.
INTRODUCTION
Nowadays, air pollution resulting from the development of industry is the most important
problem of all living creatures. Many pollutants and heavy metals are spreading rapidly
to the environment through natural, industrial and agricultural sources, municipal wastes
and atmospheric pollutants. Heavy metals are the main factors for the environment
pollution due to their toxic effects and accumulation features.
Some heavy metals such as chromium, cobalt, copper, manganese, and zinc are the
essential micronutrients for plants and animals. However, they are easily assimilated
by plants and animals and accumulated in their structures. Diagomanolin et al., [13]
and Beijer and Jernelov [3] reported that heavy metals are critical because of their easy
uptake into the food chain and bioaccumulation processes [39]. According to the studies
of Knasmuller et al., [27] and Hartwig [22], toxic heavy metals cause DNA damage and
vol. 39
no. 1
pp. 75 - 82
2013
PL ISSN 2083-4772 DOI: 10.2478/aep-2013-0007 © Copyright by Polish Academy of Sciences and Institute of Environmental Engineering of the Polish Academy of Sciences,
Zabrze, Poland 2012
INDUCTION OF MICRONUCLEI IN MICE BONE MARROW CELLS
BY COBALT AND COPPER CHLORIDES
their carcinogenic effects in animals and humans are most probably caused by their
mutagenic ability [28]. Reporting on the mutagenic activity of heavy metals by using
different assays on the genetic system of living organism is important in environmental
studies.
Different testing methods have been used to assess the genotoxicity of heavy metals
[16, 19, 21, 26, 55]. However, the mouse bone marrow micronucleus test is one of the most
widely used genetic toxicology assays. Micronuclei consist of chromosome fragments or
whole chromosomes which lag behind at anaphase of mitosis and are not incorporated
into daughter nuclei. They form single or multiple micronuclei in the cytoplasm. The
assay is based on the increase in the frequency of micronucleated PCEs in bone marrow
of the treated animals [14].
Cobalt is used for the production of alloys and hard metal (cemented carbide), in
diamond polishing, as drying agents, pigment and catalysts [9]. The available genotoxicity
data suggest that cobalt is a toxic agent [9, 33, 44]. But in recent studies, it is stated that
negative results were obtained in bacterial assays [4].
Copper is a naturally occuring metal that possesses high electrical and thermal
conductivity and resists corrosion. It is essential for human health [34]. Copper is also
an essential micronutrient for higher plant growth and metabolism [31]. However, Hall
[20], Schutzendubel and Polle [42], Yruela [56] declared that its high bioavailability in
soils makes it a potentially toxic substance causing the inhibition of growth and oxidative
injuries [49]. Several testing methods which have been used to assess the genotoxicity
of copper compounds in different test systems produced positive results [11, 18, 19, 23,
43]. However, negative results have also been reported in different test systems [32, 35,
45, 52, 54,].
Although cobalt and copper toxicities have been extensively investigated at different
test systems, there are few reports of genotoxicity in mice in vivo assays [33, 38, 40].
Because of the controversial results stated in previous studies and the widespread use
of pesticides, fertilizers, continuous air emissions from industrial sources and vehicular
traffi c throughout the world, additional studies are needed to evaluate the potential
toxic risks of these heavy metals. The aim of this study was to investigate the genotoxic
effects of the heavy metals cobalt and copper chloride by the use of mice bone marrow
micronucleus assay in vivo.
MATERIAL AND METHODS
Animals
In the present investigation, in vivo studies were carried out in 8–10 weeks old (25–30 gr)
Swiss albino mice (Mus musculus), obtained from the Labo ratuary Animal Center of
Trakya University (Edirne, Turkey). They were housed in plastic cages with a bedding
of wood shavings. They were fed fresh standart pellet and given water ad libitium. All
mice were kept under constant environmental conditions within a 12/12h ligth/dark cycle.
Chemicals, dose and treatment
Cobalt chloride (CAS No:7791-13-1) and copper chloride (CAS No: 10125-13-0) were
purchased from Merck (Whitehouse Station, NJ, USA). Each chemical was dissolved in
distilled water.
The intraperitoneal (i.p.) route of application was used in all experiments. For mice,
the LD
50concentrations (i.p.) of cobalt chloride and copper chloride were 90 mg kg
-1bw
[44] and 9.4 mg kg
-1bw [7], respectively. Cobalt chloride (45, 22.5, 11.25 mg kg
-1bw)
and copper chloride (4.70, 2.35, 1.17 mg kg
-1bw) were injected i.p. to mice for 24 and
48 hours. The lowest concentrations were 1/8 of the LD
50concentration of cobalt and
copper chloride.
Distilled water was used as a negative control. Mitomycin C (CAS No: 50-07-7)
was used as a positive control and given i.p. in a single dose of 2 mg kg
-1bw per mouse.
Micronucleus assay
In the MN test, each concentration group, negative and positive control group
contained 5 male mice. Mice (8–10 weeks) were treated with the same concentrations
intraperitoneally for 24 and 48 hours. The micronucleus test was performed according
to Schmid [41] and Aaron et al. [1] with minor modifi cations. The bone marrow cells
were fl ushed out with fetal calf serum, and the suspension was centrifuged for 10 min at
2000 rpm. The pellets were spread on a slide glass and fi xed with methanol. The slides
were stained with May-Grunwald (Cat. No. 101424) for 3 min, May Grunwald: distilled
water (1:1) for 2 min, 10% Giemsa (CAS No. 51811-82-6) in Sörensen buffer for 10 min.
A total of 1000 erythrocytes were scored for each animal at a magnifi cation of ×1000. The
numbers of MNPCE and MNNCE were also counted. PCE/NCE ratio was calculated to
determine the cytotoxic effects of the chemicals.
Statistical evaluation
The normality of the distribution of the frequency of MNPCE scores was assessed using
the non parametric Kruskal-Wallis and Mann-Whitney U test. All statistical analyses
were carried out by the Statistica programme for Windows.
RESULTS AND DISCUSSION
The results obtained are presented Table 1. Cobalt chloride induced a signifi cant increase
in the frequency of micronucleated PCE at 22.5 and 45 mg kg
-1bw concentrations for
24 hours and at all the concentrations for 48 hours when compared with the control
(Figures 1 and 2). No signifi cant reduction for the PCE/NCE was observed at all the
concentrations for 24 and 48 hours.
Copper chloride increased signifi cantly the number of micronucleated PCE at 2.35
and 4.7 mg kg
-1bw concentrations for 24 hours and all the concentrations for 48 hours
as compared with the control group (Figures 1 and 2). Also signifi cant reduction for the
PCE/NCE was observed at all the concentrations for 24 and 48 hours.
Heavy metals are a major class of environmental pollutants. Heavy metal intoxication
is a new threat issue for public health at present. There are many reports about occupational
and environmental intoxication [29, 46, 51, 53]. The detection of genotoxicity of heavy
metals is very important because people exposed to these chemicals may be adversely
affected [9, 24, 25]. So cobalt and copper chloride have been extensively investigated to
determine the damaging effects of these heavy metals.
According to the present investigation, cobalt chloride induced signifi cant
frequencies of MN in mice at different concentrations and time intervals studied. Also
Table 1. Micronucleus induction in mice bone marrow cells injected with cobalt chloride and copper chloride. T reatments Concentrations mg kg -1
Total cell number mice number Sampling time (h) Total MNPCE % PCE/NCE Sampling time (h) Total MNPCE % PCE/NCE Negative control -5000/5 24 0.70 ± 0.09 1.57 ± 0.04 48 0.67 ± 0.08 1.78 ± 0.08 Positive control 2 5000/5 4.60 ± 0.57*** 0.94 ± 0.05* 4.25 ± 0.30*** 0.85 ± 0.02* Cobalt chloride 11.2 5000/5 1.10 ± 0.19 1.43 ± 0.10 2.35 ± 0.17*** 1.70 ± 0.33 22.5 5000/5 1.45 ± 0.25* 1.45 ± 0.08 1.90 ± 0.40** 1.80 ± 0.39 45 5000/5 1.70 ± 0.02** 1.51 ± 0.01 2.55 ± 0.53*** 2.81 ± 0.38 Copper chloride 1.17 5000/5 1.05 ± 0.09 0.33 ± 0.05*** 1.30 ± 0.12* 0.29 ± 0.03*** 2.35 5000/5 1.35 ± 0.20* 0.25 ± 0.06*** 1.35 ± 0.17* 0.20 ± 0.01*** 4.7 5000/5 1.55 ± 0.30** 0.26 ± 0.03*** 2.00 ± 0.50** 0.41 ± 0.09***
MNPCE, micronucleated polychromatic erythrocyte; PCE, polychromatic erythrocyte; NCE, normochromatic erythrocyte.
All data are
presented as mean ± standart error . * P<0.05 ** P<0.01 *** P<0.001
in the studies of Sigma Aldrich [44] it is reported that cobalt chloride is mutagenic in
human lymphocytes, mammalian somatic cells. In addition, it affects the reproductive
system of mice at different concentrations. It has been reported by De Boeck et al. [9]
that cobalt (II) ions are genotoxic in vitro, and in vivo, and carcinogenic in rodents; by
De Boeck et al. [10] that cobalt caused DNA single strand breaks and micronuclei in
mammalian cells in vitro. According to NTP studies [33] cobalt (II) chloride induced
aneuploidies, micronuclei and chromosome aberrations in bone marrow in mice.
Therefore, it is stated that cobalt chloride is genotoxic in mice bone marrow cells.
These results are in agreement with our studies on the positive genotoxicity of cobalt
chloride.
In this study, copper chloride is genotoxic in mice at different concentrations tested.
Previous studies have also demonstrated that copper have mutagenic and genotoxic
activities in different biological test systems [6, 12, 18, 30, 36, 43, 47, 49, 51, 55].
Furthermore, many earlier authors [32, 35, 37, 45, 48, 50, 52, 54] have adversely observed
toxicity of copper against different test organisms. However, there are few studies
addressing the genotoxicity of copper in mice. Bhunya and Pati [5] indicated that copper
sulphate induced chromosomal aberrations, micronuclei and sperm abnormalities in
mice bone marrow cells. It has been reported that copper sulphate induced chromosomal
aberrations [2] and it was genotoxic in mice [15]. Pra et al. [38] and Saleha et al. [40]
have observed the genotoxicity and mutagenicity of copper in mice, respectively. These
statements are consistent with the results of the present study.
Several studies have shown that metal genotoxicity was caused by indirect
mechanisms. Gabbianelli et al. [17] reported that copper genotoxicity is induced
by oxidative stress and production of DNA damaging reactive oxygen species [36].
Beyersmann and Hartwig [4] also indicated that indirect mechanisms such as induction
of oxidative stress, its interference with DNA repair and deregulation of cell proliferation
cause metal genotoxicity.
Fig. 1. The increase in the frequency of micronucleated PCE observed in mice treated with cobalt chloride and copper chloride for 24 hours
PCE/NCE ratio is indicative of bone marrow cytotoxicity. A signifi cant decrease of
PCE/NCE ratio in treated animals provides evidence of an erythropoiesis depression,
with reduced proliferation of nucleated erythrocyte precursor cells [8]. Cobalt chloride
decreased PCE/NCE ratio in mice at concentrations tested when compared with the control
group. Contrary to this, copper chloride decreased PCE/NCE ratio at all concentrations in
mice for 24 and 48 hours. So it can be accepted as a cytotoxic agent.
CONCLUSIONS
The formation of MN observed at 24 and 48 hour sampling times indicated that both
cobalt and copper chloride showed their genotoxic effects. In addition, copper chloride has
cytotoxic effects in mice because it decreases PCE/NCE ratio. The in vivo micronucleus
assay used in this study was a very sensitive and reliable method to evaluate the genotoxic
effect in mammalian cells exposed to chemical substances. According to these results,
cobalt chloride and copper chloride seem to potentiate genotoxic effect in mice bone
marrow. Our observations may indicate that environment polluted with cobalt chloride
and copper chloride may lead to severe damage of human health.
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